Porphyria cutanea tarda (PCT), the most common form of porphyria in humans, is associated with excess liver iron stores, alcohol abuse, hepatitis C and medicinal estrogen use. PCT is due to a reduction in the specific activity of the heme biosynthetic enzyme uroporphyrinogen decarboxylase (URO-D) in the liver. We have determined that impaired catalytic activity of URO-D is caused by a competitive inhibitor designated uroporphomethene, an octacarboxylic tetrapyrrole macrocycle with a single oxidized bridge carbon between two of the pyrrole rings. Oxidation of the bridge carbon is mediated by P450 and iron dependent reactions. Our first specific aim is to determine if the porphomethene is generated by oxidation of hydroxymethyl bilane which then cyclizes non- enzymatically or by oxidation of uroporphyrinogen, the fully reduced substrate of URO-D. We will test the hypothesis that specific P450s and iron are required to generate the porphomethene by transfecting wild type and mutant yeast with functional human P450 systems and by altering cytosolic iron concentrations. Our second specific aim will utilize a structural approach to define the mechanism of porphomethene mediated inhibition and to confirm our model of the URO-D reaction. Our third specific aim will address the cause of iron loading in PCT. We will test the hypothesis that suppression of hepcidin expression is responsible for the excess liver iron stores. Collectively, these studies will define the molecular mechanisms involved in the pathogenesis of PCT and explain the relative rarity of PCT in face of the high incidence of disease associated risk factors. PUBLIC HEALTH RELEVANCE: Porphyria cutanea tarda (PCT) is characterized by accumulation in the liver of a compound that moves through the blood stream to the skin. The compound, called uroporphyrin, absorbs light from the sun and liberates energy particles that damage the skin. The disease is due to deficient activity of an enzyme in the liver. We have determined that deficient enzyme activity is caused by an inhibitor generated when excess iron is present in the liver. Many patients with PCT have liver damage from alcohol abuse, hepatitis C or from use of certain hormones. We propose to determine how environmental and genetic factors combine to allow the enzyme inhibitor to develop.